Andris Kazaks - Academia.edu (original) (raw)

Papers by Andris Kazaks

FEBS Journal, 2006

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR s... more The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.

Journal of Structural Biology, 2015

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from in... more Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Scientific Reports, 2015

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful prote... more Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

LU Biomedicīnas pētījumu un studiju centrs Rātsupītes iela 1, Rīga, LV-1067 E-pasts: andris@biome... more LU Biomedicīnas pētījumu un studiju centrs Rātsupītes iela 1, Rīga, LV-1067 E-pasts: andris@biomed.lu.lv Ar gēnu inženierijas metodēm tika konstruēta virkne himēru B hepatīta vīrusa nukleokapsīda proteīna (HBc) gēnu, kuri ietvēra tumorspecifiskā MAGE-3 antigēna epitopu. Ekspresējot šos gēnus Escherichia coli šūnās, tikai viens no septiņiem himērajiem HBc-MAGE proteīniem saglabāja spēju veidot vīrusveidīgas daļiņas (virus-like particles, VLP). Himērās VLP tika attīrītas no E. coli proteīniem, lai inkorporētu to sastāvā imūnstimulējošus oligonukleotīdus. Šādas struktūras varētu tikt izmantotas kā antitumoru vakcīnas. Atslēgvārdi: vīrusveidīgas daļiņas, B hepatīta vīrusa nukleokapsīds, CTL epitopi, vakcīnas.

Chemical communications (Cambridge, England), Jan 27, 2015

1-N-Alkylated-6-sulfamoyl saccharin derivatives were prepared and assayed as carbonic anhydrase i... more 1-N-Alkylated-6-sulfamoyl saccharin derivatives were prepared and assayed as carbonic anhydrase inhibitors (CAIs). During X-ray crystallographic experiments an unexpected hydrolysis of the isothiazole ring was evidenced which allowed us to prepare highly potent enzyme inhibitors with selectivity for some isoforms with medical applications.

The Journal of general virology, 2004

The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted ... more The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc-preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium- and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76-85 in the MIR, the incorporation level of HBc-preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite... more Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH.

Intervirology, 1996

Summary The Qβ gene C has been proposed as a new carrier for the exposure of foreign peptide sequ... more Summary The Qβ gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known ‘display vectors’ on the basis of coat proteins of RNA phage group I, group III phage Qβ-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called Al protein for insertion of the

Protein Expression and Purification, 2014

Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conve... more Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conversion of N6,N6,N6-trimethyl-l-lysine to 3-hydroxy-N6,N6,N6-trimethyl-l-lysine. By changing carnitine availability it is possible to optimise cardiac energy metabolism, that is beneficial under certain ischemic conditions. Previous efforts have been devoted towards the inhibition of gamma-butyrobetaine dioxygenase, which catalyses the last step in carnitine biosynthesis. However, the effects of TMLH activity regulation are currently unexplored. To facilitate the development of specific ligands of TMLH, large quantities of recombinant protein are necessary for downstream binding and structural studies. Here, we describe an efficient system for expressing and purifying active and stable TMLH as a maltose-binding protein fusion in Escherichiacoli.

The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted ... more The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc-preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium-and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76-85 in the MIR, the incorporation level of HBc-preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.

Virus Genes, 2007

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino ... more The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position -4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa -4 to aa 384; VP1(ext)). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1(ext) variant with an amino acid exchange of the authentic Met1Gly (VP1(ext-M1)) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1(ext) and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from "empty" VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.

Viral Immunology, 2002

The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural ... more The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papillomafree hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1. nomic region encoding the tumor (T) antigens and a late region coding for the capsid proteins. These regions are separated by the regulatory region containing the origin of replication and the transcriptional regulatory signals for the early and late region. The virions are non-enveloped and approximately 40 nm in diameter. The icosahedral capsid consists of three virus-encoded structural proteins: 360 molecules of the major capsid protein VP1 and 30-60 molecules of each of the minor capsid proteins VP2 and VP3. The capsid is built up by 72 pentameric capsomers in skewed (T 5 7) arrangement, each containing five molecules of VP1. The C-terminal arm of VP1 provides interactions between the capsomers. This flexible geometry of the pentamer interactions allows the formation of an icosahedral capsid (6,52). The VP1 of various polyomaviruses expressed in heterologous hosts has been demonstrated to assemble spontaneously in vitro and in vivo to virus-like particles (VLPs) .

Ticks and Tick-borne Diseases, 2014

Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host or... more Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host organisms by infected Ixodes ticks. Transfer of the spirochaetal bacteria from Ixodes ticks to the warm-blooded mammalian organism provides a challenge for the bacteria to adapt and survive in the different environmental conditions. B. burgdorferi has managed to differentially express genes in response to the encountered changes such as temperature and pH variance or metabolic rate to survive in both environments. In recent years, much interest has been turned on genes that are upregulated during the borrelial transfer to mammalian organisms as this could reveal the proteins important in the pathogenesis of Lyme disease. BBA66 is one of the upregulated outer surface proteins thought to be important in the pathogenesis of B. burgdorferi as it has been found out that BBA66 is necessary during the transmission and propagation phase to initiate Lyme disease. As there is still little known about the pathogenesis of B. burgdorferi, we have solved the crystal structure of the outer surface protein BBA66 at 2.25Å resolution. A monomer of BBA66 consists of 6 α-helices packed in a globular domain, and the overall folding is similar to the homologous proteins BBA64, BBA73, and CspA. Structure-based sequence alignment with the homologous protein BBA64 revealed that the conserved residues are mainly located inwards the core region of the protein and thus may be required to maintain the overall fold of the protein. Unlike the other homologous proteins, BBA66 has an atypically long disordered region at the N terminus thought to act as a "tether" between the structural domain and the cell surface.

FEBS Journal, 2014

Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types o... more Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types of hosts in nature - Ixodes ticks and various mammalian organisms. To initiate disease and survive in mammalian host organisms, B. burgdorferi must be able to transfer to a new host, proliferate, attach to different tissue and resist the immune response. To resist the host's immune response, B. burgdorferi produces at least five different outer surface proteins that can bind complement regulator factor H (CFH) and/or factor H-like protein 1 (CFHL-1). The crystal structures of two uniquely folded complement binding proteins, which belong to two distinct gene families and are not found in other bacteria, have been previously described. The crystal structure of the CFH and CFHL-1 binding protein CspZ (also known as BbCRASP-2 or BBH06) from B. burgdorferi, which belongs to a third gene family, is reported in this study. The structure reveals that the overall fold is different from the known structures of the other complement binding proteins in B. burgdorferi or other bacteria; this structure does not resemble the fold of any known protein deposited in the Protein Data Bank. The N-terminal part of the CspZ protein forms a four-helix bundle and has features similar to the FAT domain (focal adhesion targeting domain) and a related domain found in the vinculin/α-catenin family. By combining our findings from the crystal structure of CspZ with previous mutagenesis studies, we have identified a likely binding surface on CspZ for CFH and CFHL-1.

Protein Expression and Purification, 2011

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are rou... more Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.

Molecular Biotechnology, 2014

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the... more The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber-or opalsuppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11-16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIIIinduced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.

Molecular Biotechnology, 2014

Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have b... more Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qβ, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.

Journal of Virology, 2011

The complete genome sequence of caulobacter phage phiCb5 has been determined, and four open readi... more The complete genome sequence of caulobacter phage phiCb5 has been determined, and four open reading frames (ORFs) have been identified and characterized. As for related phages, the ORFs code for maturation, coat, replicase, and lysis proteins, but unlike other Leviviridae members, the lysis protein gene of phiCb5 entirely overlaps with the replicase in a different reading frame. The lysis protein of phiCb5 is about two times longer than that of the distantly related MS2 phage and presumably contains two transmembrane helices. Analysis of the proposed genome secondary structure revealed a stable 5 stem-loop, similar to other phages, and a substantially shorter 3 untranslated region (UTR) structure with only three stem-loops.

Journal of Molecular Biology, 2009

The structure of the Leviviridae bacteriophage phiCb5 virus-like particle has been determined at ... more The structure of the Leviviridae bacteriophage phiCb5 virus-like particle has been determined at 2.9 A resolution and the structure of the native bacteriophage phiCb5 at 3.6 A. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the phiCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, phiCb5 particles are destabilized in solution with elevated salt concentration. The model of the phiCb5 capsid provides an explanation of the salt-induced destabilization of phiCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions. Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the phiCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for phiCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The phiCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.

Journal of Medicinal Chemistry, 2013

Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (... more Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. We demonstrate that sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) possess a similar mechanism of action, acting as effective CA inhibitors. The sulfocoumarins were hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids, which thereafter bind to the enzyme in a region rarely occupied by other classes of inhibitors. The X-ray structure of one of these compounds in adduct with a modified CA II enzyme possessing two amino acid residues from the CA IX active site, allowed us to decipher the inhibition mechanism. The sulfonic acid was observed anchored to the zinc-coordinated water molecule, making favorable interactions with Thr200 and Pro201. Some other sulfocoumarins incorporating substituted-1,2,3-triazole moieties were prepared by using click chemistry and showed low nanomolar inhibitory action against the tumor-associated isoforms CA IX and XII, being less effective against the cytosolic CA I and II.

FEBS Journal, 2006

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR s... more The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.

Journal of Structural Biology, 2015

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from in... more Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Scientific Reports, 2015

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful prote... more Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

LU Biomedicīnas pētījumu un studiju centrs Rātsupītes iela 1, Rīga, LV-1067 E-pasts: andris@biome... more LU Biomedicīnas pētījumu un studiju centrs Rātsupītes iela 1, Rīga, LV-1067 E-pasts: andris@biomed.lu.lv Ar gēnu inženierijas metodēm tika konstruēta virkne himēru B hepatīta vīrusa nukleokapsīda proteīna (HBc) gēnu, kuri ietvēra tumorspecifiskā MAGE-3 antigēna epitopu. Ekspresējot šos gēnus Escherichia coli šūnās, tikai viens no septiņiem himērajiem HBc-MAGE proteīniem saglabāja spēju veidot vīrusveidīgas daļiņas (virus-like particles, VLP). Himērās VLP tika attīrītas no E. coli proteīniem, lai inkorporētu to sastāvā imūnstimulējošus oligonukleotīdus. Šādas struktūras varētu tikt izmantotas kā antitumoru vakcīnas. Atslēgvārdi: vīrusveidīgas daļiņas, B hepatīta vīrusa nukleokapsīds, CTL epitopi, vakcīnas.

Chemical communications (Cambridge, England), Jan 27, 2015

1-N-Alkylated-6-sulfamoyl saccharin derivatives were prepared and assayed as carbonic anhydrase i... more 1-N-Alkylated-6-sulfamoyl saccharin derivatives were prepared and assayed as carbonic anhydrase inhibitors (CAIs). During X-ray crystallographic experiments an unexpected hydrolysis of the isothiazole ring was evidenced which allowed us to prepare highly potent enzyme inhibitors with selectivity for some isoforms with medical applications.

The Journal of general virology, 2004

The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted ... more The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc-preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium- and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76-85 in the MIR, the incorporation level of HBc-preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite... more Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH.

Intervirology, 1996

Summary The Qβ gene C has been proposed as a new carrier for the exposure of foreign peptide sequ... more Summary The Qβ gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known ‘display vectors’ on the basis of coat proteins of RNA phage group I, group III phage Qβ-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called Al protein for insertion of the

Protein Expression and Purification, 2014

Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conve... more Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conversion of N6,N6,N6-trimethyl-l-lysine to 3-hydroxy-N6,N6,N6-trimethyl-l-lysine. By changing carnitine availability it is possible to optimise cardiac energy metabolism, that is beneficial under certain ischemic conditions. Previous efforts have been devoted towards the inhibition of gamma-butyrobetaine dioxygenase, which catalyses the last step in carnitine biosynthesis. However, the effects of TMLH activity regulation are currently unexplored. To facilitate the development of specific ligands of TMLH, large quantities of recombinant protein are necessary for downstream binding and structural studies. Here, we describe an efficient system for expressing and purifying active and stable TMLH as a maltose-binding protein fusion in Escherichiacoli.

The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted ... more The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc-preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium-and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76-85 in the MIR, the incorporation level of HBc-preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.

Virus Genes, 2007

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino ... more The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position -4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa -4 to aa 384; VP1(ext)). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1(ext) variant with an amino acid exchange of the authentic Met1Gly (VP1(ext-M1)) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1(ext) and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from "empty" VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.

Viral Immunology, 2002

The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural ... more The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papillomafree hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1. nomic region encoding the tumor (T) antigens and a late region coding for the capsid proteins. These regions are separated by the regulatory region containing the origin of replication and the transcriptional regulatory signals for the early and late region. The virions are non-enveloped and approximately 40 nm in diameter. The icosahedral capsid consists of three virus-encoded structural proteins: 360 molecules of the major capsid protein VP1 and 30-60 molecules of each of the minor capsid proteins VP2 and VP3. The capsid is built up by 72 pentameric capsomers in skewed (T 5 7) arrangement, each containing five molecules of VP1. The C-terminal arm of VP1 provides interactions between the capsomers. This flexible geometry of the pentamer interactions allows the formation of an icosahedral capsid (6,52). The VP1 of various polyomaviruses expressed in heterologous hosts has been demonstrated to assemble spontaneously in vitro and in vivo to virus-like particles (VLPs) .

Ticks and Tick-borne Diseases, 2014

Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host or... more Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host organisms by infected Ixodes ticks. Transfer of the spirochaetal bacteria from Ixodes ticks to the warm-blooded mammalian organism provides a challenge for the bacteria to adapt and survive in the different environmental conditions. B. burgdorferi has managed to differentially express genes in response to the encountered changes such as temperature and pH variance or metabolic rate to survive in both environments. In recent years, much interest has been turned on genes that are upregulated during the borrelial transfer to mammalian organisms as this could reveal the proteins important in the pathogenesis of Lyme disease. BBA66 is one of the upregulated outer surface proteins thought to be important in the pathogenesis of B. burgdorferi as it has been found out that BBA66 is necessary during the transmission and propagation phase to initiate Lyme disease. As there is still little known about the pathogenesis of B. burgdorferi, we have solved the crystal structure of the outer surface protein BBA66 at 2.25Å resolution. A monomer of BBA66 consists of 6 α-helices packed in a globular domain, and the overall folding is similar to the homologous proteins BBA64, BBA73, and CspA. Structure-based sequence alignment with the homologous protein BBA64 revealed that the conserved residues are mainly located inwards the core region of the protein and thus may be required to maintain the overall fold of the protein. Unlike the other homologous proteins, BBA66 has an atypically long disordered region at the N terminus thought to act as a "tether" between the structural domain and the cell surface.

FEBS Journal, 2014

Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types o... more Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types of hosts in nature - Ixodes ticks and various mammalian organisms. To initiate disease and survive in mammalian host organisms, B. burgdorferi must be able to transfer to a new host, proliferate, attach to different tissue and resist the immune response. To resist the host's immune response, B. burgdorferi produces at least five different outer surface proteins that can bind complement regulator factor H (CFH) and/or factor H-like protein 1 (CFHL-1). The crystal structures of two uniquely folded complement binding proteins, which belong to two distinct gene families and are not found in other bacteria, have been previously described. The crystal structure of the CFH and CFHL-1 binding protein CspZ (also known as BbCRASP-2 or BBH06) from B. burgdorferi, which belongs to a third gene family, is reported in this study. The structure reveals that the overall fold is different from the known structures of the other complement binding proteins in B. burgdorferi or other bacteria; this structure does not resemble the fold of any known protein deposited in the Protein Data Bank. The N-terminal part of the CspZ protein forms a four-helix bundle and has features similar to the FAT domain (focal adhesion targeting domain) and a related domain found in the vinculin/α-catenin family. By combining our findings from the crystal structure of CspZ with previous mutagenesis studies, we have identified a likely binding surface on CspZ for CFH and CFHL-1.

Protein Expression and Purification, 2011

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are rou... more Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.

Molecular Biotechnology, 2014

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the... more The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber-or opalsuppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11-16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIIIinduced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.

Molecular Biotechnology, 2014

Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have b... more Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qβ, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.

Journal of Virology, 2011

The complete genome sequence of caulobacter phage phiCb5 has been determined, and four open readi... more The complete genome sequence of caulobacter phage phiCb5 has been determined, and four open reading frames (ORFs) have been identified and characterized. As for related phages, the ORFs code for maturation, coat, replicase, and lysis proteins, but unlike other Leviviridae members, the lysis protein gene of phiCb5 entirely overlaps with the replicase in a different reading frame. The lysis protein of phiCb5 is about two times longer than that of the distantly related MS2 phage and presumably contains two transmembrane helices. Analysis of the proposed genome secondary structure revealed a stable 5 stem-loop, similar to other phages, and a substantially shorter 3 untranslated region (UTR) structure with only three stem-loops.

Journal of Molecular Biology, 2009

The structure of the Leviviridae bacteriophage phiCb5 virus-like particle has been determined at ... more The structure of the Leviviridae bacteriophage phiCb5 virus-like particle has been determined at 2.9 A resolution and the structure of the native bacteriophage phiCb5 at 3.6 A. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the phiCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, phiCb5 particles are destabilized in solution with elevated salt concentration. The model of the phiCb5 capsid provides an explanation of the salt-induced destabilization of phiCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions. Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the phiCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for phiCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The phiCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.

Journal of Medicinal Chemistry, 2013

Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (... more Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. We demonstrate that sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) possess a similar mechanism of action, acting as effective CA inhibitors. The sulfocoumarins were hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids, which thereafter bind to the enzyme in a region rarely occupied by other classes of inhibitors. The X-ray structure of one of these compounds in adduct with a modified CA II enzyme possessing two amino acid residues from the CA IX active site, allowed us to decipher the inhibition mechanism. The sulfonic acid was observed anchored to the zinc-coordinated water molecule, making favorable interactions with Thr200 and Pro201. Some other sulfocoumarins incorporating substituted-1,2,3-triazole moieties were prepared by using click chemistry and showed low nanomolar inhibitory action against the tumor-associated isoforms CA IX and XII, being less effective against the cytosolic CA I and II.